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Molecular Composition of the Vitreous and Aging Changes
Molecular Composition of the Vitreous and Aging Changes P N Bishop, University of Manchester, Manchester, UK ã 2010 Elsevier Ltd. All rights reserved.
Glossary Collagen – This is the most abundant protein in the body and characteristically provides tissues with shape and tensile strength. Collagen molecules are composed of three polypeptide chains (called a-chains) that assemble into a triple helix. Collagen molecules form supramolecular structures such as fibrils and sheets. Extracellular matrix – This is the material on the outside of cells that is composed of proteins, such as collagens, and carbohydrates, such as glycosaminoglycans. It provides mechanical support for cells. In addition, there is a two-way communication between cells and the extracellular matrix that regulates cellular functions, including fate, morphology, proliferation, and migration. Glycosaminoglycans – These are long chains of carbohydrate consisting of repeating disaccharide units that are found in the extracellular matrix. Because they are highly charged, they attract ions and water, and this allows them to occupy large volumes and create a swelling pressure within tissues. All glycosaminoglycans are attached to a protein core, thereby forming proteoglycans, with the exception of hyaluronan. Hyaluronan – Also called hyaluronic acid, hyaluronan is a unique glycosaminoglycan because it is not synthesized as attached to a protein core and is not sulfated. It exists as long unmodified chains of the repeating disaccharide units [b1-4 glucuronic acid-b1-3 N-acetylglucosamine]n. Posterior vitreous detachment – A collapse of the residual vitreous gel away from the inner surface of the retina as far anteriorly as the posterior border of the vitreous base. It results from a combination of vitreous liquefaction and weakening of postoral vitreoretinal adhesion. The main plane of cleavage is between the inner limiting lamina of the retina and the cortical vitreous gel, although vitreoschisis can also occur.
Introduction The vitreous humor is a highly hydrated tissue with a water content of between 98% and 99.7%. It is surrounded by, and
attached to, the retina, pars plana, and lens (Figure 1). At the vitreous base, which straddles the ora serrata, there is an unbreakable adhesion between the vitreous and peripheral retina/pars plana, but further posteriorly the adhesion is less strong and weakens with aging. When the vitreous (secondary vitreous in embryological terms) is formed, it is a gel. However, with aging, the vitreous gel gradually and inevitably liquefies. A combination of agerelated gel liquefaction and weakening of postbasal vitreoretinal adhesion eventually results in posterior vitreous detachment (PVD) in 25–30% of the population during their lifetime (Figure 2). PVD is the separation of the cortical vitreous from the inner surface of the retina, which consists of a basement membrane called the inner limiting lamina (ILL), up to the posterior border of the vitreous base. PVD plays a central role in disease processes, including rhegmatogenous retinal detachment, macular hole formation, vitreomacular traction syndrome, and proliferative diabetic retinopathy. This article will discuss the macromolecular structure of the vitreous gel and the aging changes that can eventually result in PVD.
Molecular Composition of the Vitreous Extracellular Matrix The vitreous is in essence a dilute extracellular matrix. It contains a low number of macrophage-like cells called hyalocytes. These reside in the cortical vitreous and peripheral basal vitreous (Figure 1). Extracellular matrices are composite structures containing network-forming macromolecules that possess complementary properties. There are fibrillar proteins that endow the tissue with shape, strength, flexibility, and resistance to tractional forces. Then there are charged carbohydrates, particularly glycosaminoglycans (GAGs), that attract counter-ions and water thereby providing a swelling pressure that spaces apart the fibrillar proteins, inflates the tissue and resists compressive forces. In vitreous the main fibrillar proteins are collagens and in mammalian vitreous the predominant GAG is hyaluronan (Figure 3). Vitreous Collagens The vitreous gel contains a low concentration of collagen, estimated to be approximately 300 mg ml 1 in the adult human eye. Vitreous collagen is mainly synthesized during
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Molecular Composition of the Vitreous and Aging Changes
Ciliary body
Retina
Lens
Early liquefaction
Extensive liquefaction
Partial PVD
Complete PVD
Zonules Central vitreous Vitreous base Cortical vitreous Figure 1 Anatomy of the vitreous. The vitreous is surrounded by and attached to the retina, ciliary body, and lens. The central vitreous forms the bulk of the vitreous body; in this region, the collagen fibrils are at their lowest concentration and tend to be orientated in an anterior–posterior direction. The collagen fibrils of the basal vitreous are at a higher concentration and are orientated perpendicular to the vitreous base, here they insert into the pars plana and the peripheral retina and form an unbreakable adhesion. The vitreous cortex is a thin layer (100–300 mm) that surrounds the central vitreous that has a higher concentration of collagen fibrils than the central vitreous. The anterior cortex courses from the anterior vitreous base across to the posterior surface of the lens. The posterior vitreous cortex lines the surface of the retina behind the vitreous base where it is adherent to the ILL. There is no vitreous cortex over the optic disk and it is thinned over the macula. The cortical collagen fibrils are orientated parallel with the inner retinal surface and they do not generally insert directly into the ILL. Reproduced from Le Goff, M. M., and Bishop, P. N. (2008). Adult vitreous structure and postnatal changes. Eye 22: 1214–1222, with permission of the Nature Publishing Group.
embryonic development and thereafter the rate of synthesis is low. It remains unclear how much degradation of vitreous collagen occurs, but it is likely that the rate of turnover is low and indeed it is possible that collagen fibrils that are formed during embryonic development last throughout life. Collagen molecules are composed of three polypeptide chains (a-chains) that fold into a characteristic triplehelical configuration. They also contain nontriple helical regions of varying size that are present at each end of the molecule, and in some collagens interrupt the main triplehelical region. The triple-helical fold results from the a-chains having a glycine at every third amino acid, that is, a (Gly–X–Y)n sequence where X and Y can be any amino acids, but are frequently the imino acids proline and hydroxyproline. Hydroxyproline is important in stabilizing the structure of the collagen triple helix by forming extra hydrogen bonds, while lysine and hydroxylysine residues are necessary for the formation of cross-links which stabilize the collagen fibril.
Figure 2 Age-related vitreous liquefaction and PVD. Pockets of liquefaction appear within the central vitreous and these gradually coalesce with aging. There is a concurrent weakening of postoral vitreoretinal adhesion. Eventually, these combined processes can result in PVD where the liquid vitreous dissects the residual cortical gel away from the ILL as far anteriorly as the posterior border of the vitreous base. Reproduced from Le Goff, M. M., and Bishop, P. N. (2008). Adult vitreous structure and postnatal changes. Eye 22: 1214–1222, with permission of the Nature Publishing Group.
Figure 3 Schematic representation of the cooperation between two networks responsible for the gel structure of the vitreous. A network of collagen fibrils maintains the gel state and provides the vitreous with tensile strength. A network of hyaluronan (thin black lines) fills the spaces between these collagen fibrils and provides a swelling pressure to inflate the gel and help space apart the collagen fibrils. Reproduced from Le Goff, M. M., and Bishop, P. N. (2008). Adult vitreous structure and postnatal changes. Eye 22: 1214–1222, with permission of the Nature Publishing Group.
There are at least 28 different types of collagen molecules (i.e., collagen types I – XXVIII). The commonest collagens are fibril-forming collagens, including types I, II, III and V/XI, but others such as type IV collagen form
Molecular Composition of the Vitreous and Aging Changes
sheets (in basement membranes). When fibril-forming collagens are secreted into the extracellular environment they are in a soluble precursor form, the procollagen, and have terminal extensions called N- and C-propeptides. Cleavage of these propeptides by specific enzymes produces a mature collagen with just short noncollagenous (NC) telopeptides at the ends of the triple-helical molecule; this removal (or processing) of the propeptides allows the mature collagen molecules to assemble into fibrils. In some instances the N-propeptides are not removed from fibrillar collagens, but these collagens can still participate in fibril formation and the N-propeptides are then retained on the fibril surfaces. In vitreous humor nearly all of the collagen is in thin, uniform fibrils that are about 15 nm in diameter. These fibrils are heterotypic (of mixed composition) and contain the fibril-forming collagen types II and V/XI, along with type IX collagen (Figure 4). The molecules within the fibrils are cross-linked together to make the fibrils strong and resilient. The collagen fibrils of cartilage have a similar, but not identical, composition and contain collagen types II, XI, and IX. This explains why hereditary collagenopathies such as Stickler syndrome affect both vitreous and cartilage.
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is present in some, but not all of the vitreous type II procollagen, but is absent from cartilage type II procollagen. The presence or absence of this von Willebrand type C domain is dependent on alternative splicing of exon 2 of the type II procollagen gene. Its function remains uncertain, but interestingly it binds transforming growth factor-b and bone morphogenic protein-2, so it may play a role in regulating eye development. This tissue-specific differential splicing of exon 2 explains why patients have been identified with mutations in exon 2 that have a predominantly ocular or ocular only form of Stickler syndrome.
Type II collagen
Type V/XI collagen Type V/XI collagen is a minor component (10–25%) of the heterotypic vitreous collagen fibrils. Its chain composition is probably a1(XI)2a2(V). The N-terminal domain of type V/XI collagen is not removed by processing and is retained on fibril surfaces where it may play a role in regulating fibril diameter by sterically hindering the lateral growth of the fibrils. There is evidence that a closely related collagen called type V collagen has an essential role in the initiation of collagen fibril formation in tissues where it forms heterotypic fibrils with type I collagen, so type V/XI collagen may have a similar role in initiating collagen fibril formation in the vitreous.
Collagen type II is a fibril-forming collagen that forms the bulk of the vitreous collagen fibrils, accounting for 60–75% of the collagen within the fibrils. It is composed of three identical a-chains, that is, its a-chain composition is a1(II)3. The N-propeptide contains an additional domain that
Type IX collagen Type IX collagen represents up to 25% of the collagen in the heterotypic vitreous fibrils and it is made up of three distinct a-chains, that is, a1(IX)a2(IX)a3(IX). It is not a Chondroitin sulfate glycosaminoglycan chain of type IX collagen
Type V/XI collagen
Type IX collagen
Type II collagen Figure 4 Cartoon representation of the heterotypic collagen fibrils of the vitreous. The fibrils contain collagen types II, V/XI, and IX. Collagen types II and V/XI are fibril-forming collagens that align in staggered arrays to form a central core to the fibrils. Type IX collagen is a chondroitin sulfate proteoglycan that is regularly distributed along the surface of the fibrils. The N-propeptides of type V/XI collagen are retained and located on the surface of the fibrils and some of the N-propeptides of type II collagen may be retained on the fibril surfaces. Reproduced from Bishop, P. (1996). The biochemical structure of mammalian vitreous. Eye 10: 664–670, with permission of Macmillan Publishers Ltd.
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Molecular Composition of the Vitreous and Aging Changes
fibril-forming collagen. Instead it is regularly cross-linked to the surface of the collagen fibrils, which have a core formed by the type II and V/XI collagens. It is a member of the family of fibril-associated collagens with interrupted triple helices (FACIT), which also includes collagen types XII, XIV, and XVI. These collagens have (NC) domains interspersed between collagenous (COL) regions. In the case of type IX collagen there are three COL domains (COL1, COL2, and COL3) interspersed between four NC domains (NC1, NC2, NC3, and NC4). Further complexity is added by the a1(IX) collagen gene (COL9A1) using tissue-specific alternatively spliced transcription start sites, resulting in a form in cartilage that has a globular NC4 domain, but this domain is almost absent in vitreous. The functional significance of these differences remains unclear. Type IX collagen can exist in proteoglycan and nonproteoglycan forms with the proteoglycan form having a single chondroitin sulfate chain attached to the a2(IX) chain in the NC3 domain. In vitreous, the type IX collagen is synthesized in a proteoglycan form, but the length and sulfation pattern of the chondroitin sulfate chain is species dependent. In mammalian vitreous it has a relatively short chondroitin sulfate chain (15–60 kDa), but in chick vitreous it is very long (approximately 350 kDa). Synthesis of vitreous collagen
Evidence from a variety of studies suggests that during embryonic development vitreous collagens are mainly synthesized by the ciliary body. The posterior nonpigmented ciliary epithelium secretes the collagen molecules that assemble into fibrils in or around these cells. The fibrils are then extruded into the vitreous cavity. This provides an explanation for the perpendicular orientation of the collagen fibrils in the basal vitreous relative to the nonpigmented ciliary epithelium, and the unbreakable adhesion at the vitreous base. While the levels are low, there is good evidence that some postnatal vitreous collagen synthesis occurs. Interestingly, this appears to carry on into adulthood and to take place in the peripheral retina. The cells responsible for collagen synthesis in the adult eye are yet to be conclusively identified. This new vitreous collagen synthesis in the adult eye results in the formation of a mat of collagen fibrils on the cellular side of the peripheral ILL. Some of these collagen fibrils break through the ILL and become intertwined with the preexisting cortical vitreous collagen. This process then results in the formation of new unbreakable vitreoretinal adhesions and thereby extends posteriorly at the posterior border of the vitreous base (Figure 5). At birth, the vitreous base is at the ora serrata, but it extends posteriorly with aging and has been shown to have migrated over 3.5 mm behind the ora serrata as a result of this process. Irregularities of the posterior border of the vitreous base resulting from uneven posterior extension will predispose to rhegmatogenous retinal detachment during PVD.
ILL
Müller cell footplates
Figure 5 Extension of the posterior border of the vitreous base. There is a very strong adhesion at the vitreoretinal interface within the vitreous base because vitreous collagen fibrils insert directly into the posterior ciliary body and peripheral retina. The vitreous base extends posteriorly into the peripheral retina with aging as a result of the adult peripheral retina synthesizing new collagen. This new collagen forms a mat-like layer on the cellular side of the ILL, and some of this collagen breaks through defects in the ILL and intertwines with preexisting cortical vitreous collagen thereby creating new adhesions and extending the vitreous base posteriorly. Reproduced from Le Goff, M. M., and Bishop, P. N. (2008). Adult vitreous structure and postnatal changes. Eye 22: 1214–1222, with permission of the Nature Publishing Group.
NC Proteins and Glycoproteins Opticin The heterotypic collagen fibrils of the vitreous are coated with a molecule called opticin. It is a glycoprotein member of the extracellular matrix small leucine-rich repeat protein/proteoglycan (SLRP) family. This family comprises of approximately 11 members and are characterized by a domain containing a number of tandem leucine-rich repeats (LRRs) flanked by disulfide-bonded capping motifs. LRRs are 20–30 amino acid repeats that contain the consensus sequence LXXLXLXXNXL, where L can be a leucine, isoleucine, or valine residue, N is an asparagine, cysteine, or threonine residue and X can be any amino acid. Opticin possesses eight tandem LRRs and an N-terminal extension containing a cluster of sialylated O-linked oligosaccharides. Opticin exists as a dimer in solution as a result of interactions between the LRR domains. Other members of the SLRP family have been shown to play a role in regulating collagen fibril diameter, but this does not appear to be the case for opticin. Instead, opticin appears to modify the vitreous collagen fibril surfaces that are presented to invading cells during pathological processes, such as preretinal neovascularization, and thereby it can attenuate disease processes.
Molecular Composition of the Vitreous and Aging Changes
Other proteins and glycoproteins
Vitreous contains many other proteins. Some are derived from the serum and others are produced locally. As well as the molecules described above there are other extracellular matrix components in the vitreous, but their functions remain uncertain. Fibrillin-containing microfibrils have been demonstrated in vitreous. The zonules are composed of bundles of these fibrils and it is unclear whether the vitreous fibrillin-containing microfibrils have a structural role or whether they are a byproduct of zonular synthesis or breakdown. A small amount of type VI collagen is present, this forms its own distinctive microfibrils, but again it is unclear if these have a significant structural role. Fibronectin has been identified in vitreous and it has been postulated that it is involved in vitreoretinal adhesion. A number of other extracellular matrix components have been identified using proteomics in embryonic chick vitreous.
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remains uncertain. Nonetheless, it is of note that the hereditary vitreoretinopathy Wagner syndrome is caused by splice site mutations in versican. Heparan sulfate proteoglycans Heparan sulfate and heparin share the same repeating disaccharide unit [b1-4 glucuronic acid-a1-4 N-acetylglucosamine]n. The GAG chain is variably modified by sulfation, deacetylation, and epimerization. The degree of modification determines whether the GAG is heparan sulfate or heparin. Heparan sulfate proteoglycans are major components of basement membranes, including the ILL. Heparan sulfate proteoglycans are present in the embryonic vitreous, possibly in transit to form the ILL, but the levels are very low in postnatal eyes.
Supramolecular Organization of the Vitreous
GAGs and Proteoglycans GAGs are long chains of repeating disaccharide units that undergo varying degrees of modifications during their synthesis, including sulfation, epimerization, and acetylation/deacetylation. These modifications alter the charge of the GAGs and can create specific binding sites for other molecules. All GAGs are attached to a core protein, thereby forming proteoglycans, except hyaluronan which is uniquely synthesized at cell surfaces and is not linked to a core protein. Hyaluronan
Hyaluronan is the predominant GAG in mammalian vitreous. It is composed of very long carbohydrate chains consisting of the repeating disaccharide [b1-4 glucuronic acid-b1-3 N-acetylglucosamine]n. These chains are not modified by sulfation or epimerization. In adult human vitreous, the hyaluronan concentration has been estimated to be between 65 and 400 mg ml 1 and the average molecular weight to be 2–4 million. Chondroitin sulfate proteoglycans
The repeating disaccharide unit of chondroitin sulfate is [b1-4 glucuronic acid-b1-3 N-acetylgalactosamine]n. The C-4 and C-6 of the N-acetylgalactosamine residues are variably sulfated, and less frequently the C-2 of glucuronic acid. Vitreous is known to contain two chondroitin sulfate proteoglycans, type IX collagen (see section on collagens) and versican. Versican is a large proteoglycan with a central domain that carries multiple chondroitin sulfate chains. At its N-terminus it has a hyaluronanbinding domain that presumably binds vitreous hyaluronan and this binding is stabilized by a link protein. However, as the hyaluronan is in a 150:1 molar excess to the versican and link protein, the structural role of versican in vitreous
The vitreous collagen fibrils and hyaluronan form two interwoven networks (Figure 3). The network of (heterotypic) collagen fibrils is essential for the gel state of the vitreous, as removal of these collagen fibrils converts the vitreous into a viscous liquid. Furthermore, it is through this network of collagen fibrils that tractional forces are transmitted in vitreoretinal diseases. The hyaluronan network inflates the vitreous, increases its mechanical stability and contributes to the spacing of the collagen network, but surprisingly the hyaluronan network can be removed without destroying the vitreous gel, at least in the short term. The long hyaluronan chains form a network primarily through entanglement and this network probably interacts weakly with the collagen fibrillar network. The vitreous collagen fibrils are long and unbranched. Analysis by freeze-etch rotary shadowing electron microscopy showed that the collagen fibrils are arranged in bundles within the vitreous and form an extended interconnected network by branching between these bundles (Figure 6(a)). In the young eye the vitreous collagen fibrils within the bundles appear to run closely together and in parallel, but are not fused. Morphological analyses suggest that the chondroitin sulfate chains of type IX collagen play a role in both connecting together and spacing apart the collagen fibrils within these bundles (Figure 7).
Aging Changes that Predispose to PVD Age-Related Vitreous Liquefaction The human vitreous humor undergoes an inevitable process of liquefaction (or syneresis) with aging. Initially
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Molecular Composition of the Vitreous and Aging Changes
small pockets of liquefaction form, but these coalesce with aging to produce more extensive areas; so by the age of 80–90 years typically more than half of the vitreous cavity is filled with liquid. Ultrastructural studies have shown that vitreous collagen fibrils aggregate with aging into macroscopic strands. Age-related vitreous liquefaction is probably caused by this gradual and progressive aggregation of the collagen fibrils resulting in a redistribution of the collagen fibrils, with the aggregated collagen fibrils being concentrated in the residual gel and other areas therefore becoming devoid of collagen fibrils and consequently converted into liquid. The spacing between the collagen fibrils appears to be maintained by the chondroitin sulfate chains of type IX collagen (Figures 6 and 7). However, we have shown that
(a)
(b)
Figure 6 The collagen fibrillar network of the vitreous and aging changes. (a) The collagen fibrils (thick gray lines) form an extended network by being organized into small bundles that are interconnected by collagen fibrils running from one bundle to another. Within each bundle the collagen fibrils are both connected together and spaced apart by the chondroitin sulfate chains of type IX collagen (thin black lines). (b) With aging there is a loss of type IX collagen from the fibril surfaces. The loss of the type IX collagen from the fibril surfaces combined with an increased surface exposure of type II collagen results in aggregation of the collagen fibrils. Reproduced from Le Goff, M. M., and Bishop, P. N. (2008). Adult vitreous structure and postnatal changes. Eye 22: 1214–1222, with permission of the Nature Publishing Group.
there is a progressive loss of type IX collagen from the surface of the human vitreous collagen fibrils with aging, and that the half-life of type IX collagen on the fibril surfaces is just 11 years of age. This loss of type IX collagen (along with its chondroitin sulfate chains) will result in a loss of spacing between the collagen fibrils. Furthermore, the loss of type IX collagen results in the increased exposure of sticky type II collagen on the fibril surfaces, so when the collagen fibrils come into contact (as a result of eye movement) they will have a propensity to fuse irreversibly.
Weakening of the Vitreoretinal Adhesion In young eyes there is a relatively strong adhesion between the cortical vitreous and the ILL of the postoral retina, but this weakens with age. The cortical vitreous collagen fibrils do not generally insert directly into the postoral ILL, so it is assumed that the molecular basis of the vitreoretinal adhesion is interactions between components on the surface of cortical vitreous collagen fibrils and macromolecules on the inner surface of the ILL. There is evidence that type XVIII collagen is involved in vitreoretinal adhesion from studies of knockout mice and early PVD in patients with Knobloch syndrome, a rare autosomal recessive condition that results in a lack of type XVIII collagen. Type XVIII collagen is a heparan sulfate proteoglycan. It has been shown that opticin can bind heparan sulfates including those of type XVIII collagen and opticin and type XVIII collagen colocalize at the vitreoretinal interface. Therefore, the opticin on the surface of cortical vitreous collagen fibrils could bind heparan sulfate proteoglycans of the ILL including type XVIII collagen, thereby contributing to vitreoretinal adhesion. However, opticin knockout mice do not appear to develop spontaneous vitreoretinal disinsertion, suggesting that other molecular interactions also contribute to vitreoretinal adhesion, at least in the mouse eye.
Conclusions
Figure 7 The role of the chondroitin sulfate chains of type IX collagen in maintaining the spacing between vitreous collagen fibrils. Electron micrographs of vitreous collagen fibrils stained with uranyl acetate (to visualize the collagen fibrils) and the cationic dye Alcian blue (to visualize the chondroitin sulfate chains of type IX collagen). The Alcian blue-stained chondroitin sulfate chains appear to bridge between adjacent collagen fibrils and thereby both space the collagen fibrils apart and link them together. (Scale bar 300 nm).
Although the vitreous only contains a dilute dispersion of collagen fibrils, it is these fibrils that are central to the gel structure of the vitreous. Molecules on the surface of these collagen fibrils are essential for maintaining the gel state (such as type IX collagen) and play a key role in vitreoretinal adhesion. Alterations in the molecular interactions of the collagen fibrils lead to vitreous liquefaction, weakening of vitreoretinal adhesion, and a consequent predisposition to PVD. See also: Cellular Origin, Formation and Turnover of the Vitreous; Hereditary Vitreoretinopathies; Hyalocytes.
Molecular Composition of the Vitreous and Aging Changes
Further Reading Bishop, P. N. (2000). Structural macromolecules and supramolecular organisation of the vitreous gel. Progress in Retinal and Eye Research 19: 323–344. Bishop, P. N., Holmes, D. F., Kadler, K. E., McLeod, D., and Bos, K. J. (2004). Age-related changes on the surface of vitreous collagen fibrils. Investigative Ophthalmology and Visual Science 45: 1041–1046.
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Bos, K. J., Holmes, D. F., Meadows, R. S., et al. (2001). Collagen fibril organisation in mammalian vitreous by freeze etch/rotary shadowing electron microscopy. Micron 32: 301–306. Fukai, N., Eklund, L., Marneros, A. G., et al. (2002). Lack of collagen XVIII/endostatin results in eye abnormalities. EMBO Journal 21: 1535–1544. Wang, J., McLeod, D., Henson, D. B., and Bishop, P. N. (2003). Agedependent changes in the basal retinovitreal adhesion. Investigative Ophthalmology and Visual Science 44: 1793–1800.
Glossary Collagen – This is the most abundant protein in the body and characteristically provides tissues with shape and tensile strength. Collagen molecules are composed of three polypeptide chains (called a-chains) that assemble into a triple helix. Collagen molecules form supramolecular structures such as fibrils and sheets. Extracellular matrix – This is the material on the outside of cells that is composed of proteins, such as collagens, and carbohydrates, such as glycosaminoglycans. It provides mechanical support for cells. In addition, there is a two-way communication between cells and the extracellular matrix that regulates cellular functions, including fate, morphology, proliferation, and migration. Glycosaminoglycans – These are long chains of carbohydrate consisting of repeating disaccharide units that are found in the extracellular matrix. Because they are highly charged, they attract ions and water, and this allows them to occupy large volumes and create a swelling pressure within tissues. All glycosaminoglycans are attached to a protein core, thereby forming proteoglycans, with the exception of hyaluronan. Hyaluronan – Also called hyaluronic acid, hyaluronan is a unique glycosaminoglycan because it is not synthesized as attached to a protein core and is not sulfated. It exists as long unmodified chains of the repeating disaccharide units [b1-4 glucuronic acid-b1-3 N-acetylglucosamine]n. Posterior vitreous detachment – A collapse of the residual vitreous gel away from the inner surface of the retina as far anteriorly as the posterior border of the vitreous base. It results from a combination of vitreous liquefaction and weakening of postoral vitreoretinal adhesion. The main plane of cleavage is between the inner limiting lamina of the retina and the cortical vitreous gel, although vitreoschisis can also occur.
Introduction The vitreous humor is a highly hydrated tissue with a water content of between 98% and 99.7%. It is surrounded by, and
attached to, the retina, pars plana, and lens (Figure 1). At the vitreous base, which straddles the ora serrata, there is an unbreakable adhesion between the vitreous and peripheral retina/pars plana, but further posteriorly the adhesion is less strong and weakens with aging. When the vitreous (secondary vitreous in embryological terms) is formed, it is a gel. However, with aging, the vitreous gel gradually and inevitably liquefies. A combination of agerelated gel liquefaction and weakening of postbasal vitreoretinal adhesion eventually results in posterior vitreous detachment (PVD) in 25–30% of the population during their lifetime (Figure 2). PVD is the separation of the cortical vitreous from the inner surface of the retina, which consists of a basement membrane called the inner limiting lamina (ILL), up to the posterior border of the vitreous base. PVD plays a central role in disease processes, including rhegmatogenous retinal detachment, macular hole formation, vitreomacular traction syndrome, and proliferative diabetic retinopathy. This article will discuss the macromolecular structure of the vitreous gel and the aging changes that can eventually result in PVD.
Molecular Composition of the Vitreous Extracellular Matrix The vitreous is in essence a dilute extracellular matrix. It contains a low number of macrophage-like cells called hyalocytes. These reside in the cortical vitreous and peripheral basal vitreous (Figure 1). Extracellular matrices are composite structures containing network-forming macromolecules that possess complementary properties. There are fibrillar proteins that endow the tissue with shape, strength, flexibility, and resistance to tractional forces. Then there are charged carbohydrates, particularly glycosaminoglycans (GAGs), that attract counter-ions and water thereby providing a swelling pressure that spaces apart the fibrillar proteins, inflates the tissue and resists compressive forces. In vitreous the main fibrillar proteins are collagens and in mammalian vitreous the predominant GAG is hyaluronan (Figure 3). Vitreous Collagens The vitreous gel contains a low concentration of collagen, estimated to be approximately 300 mg ml 1 in the adult human eye. Vitreous collagen is mainly synthesized during
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Molecular Composition of the Vitreous and Aging Changes
Ciliary body
Retina
Lens
Early liquefaction
Extensive liquefaction
Partial PVD
Complete PVD
Zonules Central vitreous Vitreous base Cortical vitreous Figure 1 Anatomy of the vitreous. The vitreous is surrounded by and attached to the retina, ciliary body, and lens. The central vitreous forms the bulk of the vitreous body; in this region, the collagen fibrils are at their lowest concentration and tend to be orientated in an anterior–posterior direction. The collagen fibrils of the basal vitreous are at a higher concentration and are orientated perpendicular to the vitreous base, here they insert into the pars plana and the peripheral retina and form an unbreakable adhesion. The vitreous cortex is a thin layer (100–300 mm) that surrounds the central vitreous that has a higher concentration of collagen fibrils than the central vitreous. The anterior cortex courses from the anterior vitreous base across to the posterior surface of the lens. The posterior vitreous cortex lines the surface of the retina behind the vitreous base where it is adherent to the ILL. There is no vitreous cortex over the optic disk and it is thinned over the macula. The cortical collagen fibrils are orientated parallel with the inner retinal surface and they do not generally insert directly into the ILL. Reproduced from Le Goff, M. M., and Bishop, P. N. (2008). Adult vitreous structure and postnatal changes. Eye 22: 1214–1222, with permission of the Nature Publishing Group.
embryonic development and thereafter the rate of synthesis is low. It remains unclear how much degradation of vitreous collagen occurs, but it is likely that the rate of turnover is low and indeed it is possible that collagen fibrils that are formed during embryonic development last throughout life. Collagen molecules are composed of three polypeptide chains (a-chains) that fold into a characteristic triplehelical configuration. They also contain nontriple helical regions of varying size that are present at each end of the molecule, and in some collagens interrupt the main triplehelical region. The triple-helical fold results from the a-chains having a glycine at every third amino acid, that is, a (Gly–X–Y)n sequence where X and Y can be any amino acids, but are frequently the imino acids proline and hydroxyproline. Hydroxyproline is important in stabilizing the structure of the collagen triple helix by forming extra hydrogen bonds, while lysine and hydroxylysine residues are necessary for the formation of cross-links which stabilize the collagen fibril.
Figure 2 Age-related vitreous liquefaction and PVD. Pockets of liquefaction appear within the central vitreous and these gradually coalesce with aging. There is a concurrent weakening of postoral vitreoretinal adhesion. Eventually, these combined processes can result in PVD where the liquid vitreous dissects the residual cortical gel away from the ILL as far anteriorly as the posterior border of the vitreous base. Reproduced from Le Goff, M. M., and Bishop, P. N. (2008). Adult vitreous structure and postnatal changes. Eye 22: 1214–1222, with permission of the Nature Publishing Group.
Figure 3 Schematic representation of the cooperation between two networks responsible for the gel structure of the vitreous. A network of collagen fibrils maintains the gel state and provides the vitreous with tensile strength. A network of hyaluronan (thin black lines) fills the spaces between these collagen fibrils and provides a swelling pressure to inflate the gel and help space apart the collagen fibrils. Reproduced from Le Goff, M. M., and Bishop, P. N. (2008). Adult vitreous structure and postnatal changes. Eye 22: 1214–1222, with permission of the Nature Publishing Group.
There are at least 28 different types of collagen molecules (i.e., collagen types I – XXVIII). The commonest collagens are fibril-forming collagens, including types I, II, III and V/XI, but others such as type IV collagen form
Molecular Composition of the Vitreous and Aging Changes
sheets (in basement membranes). When fibril-forming collagens are secreted into the extracellular environment they are in a soluble precursor form, the procollagen, and have terminal extensions called N- and C-propeptides. Cleavage of these propeptides by specific enzymes produces a mature collagen with just short noncollagenous (NC) telopeptides at the ends of the triple-helical molecule; this removal (or processing) of the propeptides allows the mature collagen molecules to assemble into fibrils. In some instances the N-propeptides are not removed from fibrillar collagens, but these collagens can still participate in fibril formation and the N-propeptides are then retained on the fibril surfaces. In vitreous humor nearly all of the collagen is in thin, uniform fibrils that are about 15 nm in diameter. These fibrils are heterotypic (of mixed composition) and contain the fibril-forming collagen types II and V/XI, along with type IX collagen (Figure 4). The molecules within the fibrils are cross-linked together to make the fibrils strong and resilient. The collagen fibrils of cartilage have a similar, but not identical, composition and contain collagen types II, XI, and IX. This explains why hereditary collagenopathies such as Stickler syndrome affect both vitreous and cartilage.
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is present in some, but not all of the vitreous type II procollagen, but is absent from cartilage type II procollagen. The presence or absence of this von Willebrand type C domain is dependent on alternative splicing of exon 2 of the type II procollagen gene. Its function remains uncertain, but interestingly it binds transforming growth factor-b and bone morphogenic protein-2, so it may play a role in regulating eye development. This tissue-specific differential splicing of exon 2 explains why patients have been identified with mutations in exon 2 that have a predominantly ocular or ocular only form of Stickler syndrome.
Type II collagen
Type V/XI collagen Type V/XI collagen is a minor component (10–25%) of the heterotypic vitreous collagen fibrils. Its chain composition is probably a1(XI)2a2(V). The N-terminal domain of type V/XI collagen is not removed by processing and is retained on fibril surfaces where it may play a role in regulating fibril diameter by sterically hindering the lateral growth of the fibrils. There is evidence that a closely related collagen called type V collagen has an essential role in the initiation of collagen fibril formation in tissues where it forms heterotypic fibrils with type I collagen, so type V/XI collagen may have a similar role in initiating collagen fibril formation in the vitreous.
Collagen type II is a fibril-forming collagen that forms the bulk of the vitreous collagen fibrils, accounting for 60–75% of the collagen within the fibrils. It is composed of three identical a-chains, that is, its a-chain composition is a1(II)3. The N-propeptide contains an additional domain that
Type IX collagen Type IX collagen represents up to 25% of the collagen in the heterotypic vitreous fibrils and it is made up of three distinct a-chains, that is, a1(IX)a2(IX)a3(IX). It is not a Chondroitin sulfate glycosaminoglycan chain of type IX collagen
Type V/XI collagen
Type IX collagen
Type II collagen Figure 4 Cartoon representation of the heterotypic collagen fibrils of the vitreous. The fibrils contain collagen types II, V/XI, and IX. Collagen types II and V/XI are fibril-forming collagens that align in staggered arrays to form a central core to the fibrils. Type IX collagen is a chondroitin sulfate proteoglycan that is regularly distributed along the surface of the fibrils. The N-propeptides of type V/XI collagen are retained and located on the surface of the fibrils and some of the N-propeptides of type II collagen may be retained on the fibril surfaces. Reproduced from Bishop, P. (1996). The biochemical structure of mammalian vitreous. Eye 10: 664–670, with permission of Macmillan Publishers Ltd.
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Molecular Composition of the Vitreous and Aging Changes
fibril-forming collagen. Instead it is regularly cross-linked to the surface of the collagen fibrils, which have a core formed by the type II and V/XI collagens. It is a member of the family of fibril-associated collagens with interrupted triple helices (FACIT), which also includes collagen types XII, XIV, and XVI. These collagens have (NC) domains interspersed between collagenous (COL) regions. In the case of type IX collagen there are three COL domains (COL1, COL2, and COL3) interspersed between four NC domains (NC1, NC2, NC3, and NC4). Further complexity is added by the a1(IX) collagen gene (COL9A1) using tissue-specific alternatively spliced transcription start sites, resulting in a form in cartilage that has a globular NC4 domain, but this domain is almost absent in vitreous. The functional significance of these differences remains unclear. Type IX collagen can exist in proteoglycan and nonproteoglycan forms with the proteoglycan form having a single chondroitin sulfate chain attached to the a2(IX) chain in the NC3 domain. In vitreous, the type IX collagen is synthesized in a proteoglycan form, but the length and sulfation pattern of the chondroitin sulfate chain is species dependent. In mammalian vitreous it has a relatively short chondroitin sulfate chain (15–60 kDa), but in chick vitreous it is very long (approximately 350 kDa). Synthesis of vitreous collagen
Evidence from a variety of studies suggests that during embryonic development vitreous collagens are mainly synthesized by the ciliary body. The posterior nonpigmented ciliary epithelium secretes the collagen molecules that assemble into fibrils in or around these cells. The fibrils are then extruded into the vitreous cavity. This provides an explanation for the perpendicular orientation of the collagen fibrils in the basal vitreous relative to the nonpigmented ciliary epithelium, and the unbreakable adhesion at the vitreous base. While the levels are low, there is good evidence that some postnatal vitreous collagen synthesis occurs. Interestingly, this appears to carry on into adulthood and to take place in the peripheral retina. The cells responsible for collagen synthesis in the adult eye are yet to be conclusively identified. This new vitreous collagen synthesis in the adult eye results in the formation of a mat of collagen fibrils on the cellular side of the peripheral ILL. Some of these collagen fibrils break through the ILL and become intertwined with the preexisting cortical vitreous collagen. This process then results in the formation of new unbreakable vitreoretinal adhesions and thereby extends posteriorly at the posterior border of the vitreous base (Figure 5). At birth, the vitreous base is at the ora serrata, but it extends posteriorly with aging and has been shown to have migrated over 3.5 mm behind the ora serrata as a result of this process. Irregularities of the posterior border of the vitreous base resulting from uneven posterior extension will predispose to rhegmatogenous retinal detachment during PVD.
ILL
Müller cell footplates
Figure 5 Extension of the posterior border of the vitreous base. There is a very strong adhesion at the vitreoretinal interface within the vitreous base because vitreous collagen fibrils insert directly into the posterior ciliary body and peripheral retina. The vitreous base extends posteriorly into the peripheral retina with aging as a result of the adult peripheral retina synthesizing new collagen. This new collagen forms a mat-like layer on the cellular side of the ILL, and some of this collagen breaks through defects in the ILL and intertwines with preexisting cortical vitreous collagen thereby creating new adhesions and extending the vitreous base posteriorly. Reproduced from Le Goff, M. M., and Bishop, P. N. (2008). Adult vitreous structure and postnatal changes. Eye 22: 1214–1222, with permission of the Nature Publishing Group.
NC Proteins and Glycoproteins Opticin The heterotypic collagen fibrils of the vitreous are coated with a molecule called opticin. It is a glycoprotein member of the extracellular matrix small leucine-rich repeat protein/proteoglycan (SLRP) family. This family comprises of approximately 11 members and are characterized by a domain containing a number of tandem leucine-rich repeats (LRRs) flanked by disulfide-bonded capping motifs. LRRs are 20–30 amino acid repeats that contain the consensus sequence LXXLXLXXNXL, where L can be a leucine, isoleucine, or valine residue, N is an asparagine, cysteine, or threonine residue and X can be any amino acid. Opticin possesses eight tandem LRRs and an N-terminal extension containing a cluster of sialylated O-linked oligosaccharides. Opticin exists as a dimer in solution as a result of interactions between the LRR domains. Other members of the SLRP family have been shown to play a role in regulating collagen fibril diameter, but this does not appear to be the case for opticin. Instead, opticin appears to modify the vitreous collagen fibril surfaces that are presented to invading cells during pathological processes, such as preretinal neovascularization, and thereby it can attenuate disease processes.
Molecular Composition of the Vitreous and Aging Changes
Other proteins and glycoproteins
Vitreous contains many other proteins. Some are derived from the serum and others are produced locally. As well as the molecules described above there are other extracellular matrix components in the vitreous, but their functions remain uncertain. Fibrillin-containing microfibrils have been demonstrated in vitreous. The zonules are composed of bundles of these fibrils and it is unclear whether the vitreous fibrillin-containing microfibrils have a structural role or whether they are a byproduct of zonular synthesis or breakdown. A small amount of type VI collagen is present, this forms its own distinctive microfibrils, but again it is unclear if these have a significant structural role. Fibronectin has been identified in vitreous and it has been postulated that it is involved in vitreoretinal adhesion. A number of other extracellular matrix components have been identified using proteomics in embryonic chick vitreous.
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remains uncertain. Nonetheless, it is of note that the hereditary vitreoretinopathy Wagner syndrome is caused by splice site mutations in versican. Heparan sulfate proteoglycans Heparan sulfate and heparin share the same repeating disaccharide unit [b1-4 glucuronic acid-a1-4 N-acetylglucosamine]n. The GAG chain is variably modified by sulfation, deacetylation, and epimerization. The degree of modification determines whether the GAG is heparan sulfate or heparin. Heparan sulfate proteoglycans are major components of basement membranes, including the ILL. Heparan sulfate proteoglycans are present in the embryonic vitreous, possibly in transit to form the ILL, but the levels are very low in postnatal eyes.
Supramolecular Organization of the Vitreous
GAGs and Proteoglycans GAGs are long chains of repeating disaccharide units that undergo varying degrees of modifications during their synthesis, including sulfation, epimerization, and acetylation/deacetylation. These modifications alter the charge of the GAGs and can create specific binding sites for other molecules. All GAGs are attached to a core protein, thereby forming proteoglycans, except hyaluronan which is uniquely synthesized at cell surfaces and is not linked to a core protein. Hyaluronan
Hyaluronan is the predominant GAG in mammalian vitreous. It is composed of very long carbohydrate chains consisting of the repeating disaccharide [b1-4 glucuronic acid-b1-3 N-acetylglucosamine]n. These chains are not modified by sulfation or epimerization. In adult human vitreous, the hyaluronan concentration has been estimated to be between 65 and 400 mg ml 1 and the average molecular weight to be 2–4 million. Chondroitin sulfate proteoglycans
The repeating disaccharide unit of chondroitin sulfate is [b1-4 glucuronic acid-b1-3 N-acetylgalactosamine]n. The C-4 and C-6 of the N-acetylgalactosamine residues are variably sulfated, and less frequently the C-2 of glucuronic acid. Vitreous is known to contain two chondroitin sulfate proteoglycans, type IX collagen (see section on collagens) and versican. Versican is a large proteoglycan with a central domain that carries multiple chondroitin sulfate chains. At its N-terminus it has a hyaluronanbinding domain that presumably binds vitreous hyaluronan and this binding is stabilized by a link protein. However, as the hyaluronan is in a 150:1 molar excess to the versican and link protein, the structural role of versican in vitreous
The vitreous collagen fibrils and hyaluronan form two interwoven networks (Figure 3). The network of (heterotypic) collagen fibrils is essential for the gel state of the vitreous, as removal of these collagen fibrils converts the vitreous into a viscous liquid. Furthermore, it is through this network of collagen fibrils that tractional forces are transmitted in vitreoretinal diseases. The hyaluronan network inflates the vitreous, increases its mechanical stability and contributes to the spacing of the collagen network, but surprisingly the hyaluronan network can be removed without destroying the vitreous gel, at least in the short term. The long hyaluronan chains form a network primarily through entanglement and this network probably interacts weakly with the collagen fibrillar network. The vitreous collagen fibrils are long and unbranched. Analysis by freeze-etch rotary shadowing electron microscopy showed that the collagen fibrils are arranged in bundles within the vitreous and form an extended interconnected network by branching between these bundles (Figure 6(a)). In the young eye the vitreous collagen fibrils within the bundles appear to run closely together and in parallel, but are not fused. Morphological analyses suggest that the chondroitin sulfate chains of type IX collagen play a role in both connecting together and spacing apart the collagen fibrils within these bundles (Figure 7).
Aging Changes that Predispose to PVD Age-Related Vitreous Liquefaction The human vitreous humor undergoes an inevitable process of liquefaction (or syneresis) with aging. Initially
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Molecular Composition of the Vitreous and Aging Changes
small pockets of liquefaction form, but these coalesce with aging to produce more extensive areas; so by the age of 80–90 years typically more than half of the vitreous cavity is filled with liquid. Ultrastructural studies have shown that vitreous collagen fibrils aggregate with aging into macroscopic strands. Age-related vitreous liquefaction is probably caused by this gradual and progressive aggregation of the collagen fibrils resulting in a redistribution of the collagen fibrils, with the aggregated collagen fibrils being concentrated in the residual gel and other areas therefore becoming devoid of collagen fibrils and consequently converted into liquid. The spacing between the collagen fibrils appears to be maintained by the chondroitin sulfate chains of type IX collagen (Figures 6 and 7). However, we have shown that
(a)
(b)
Figure 6 The collagen fibrillar network of the vitreous and aging changes. (a) The collagen fibrils (thick gray lines) form an extended network by being organized into small bundles that are interconnected by collagen fibrils running from one bundle to another. Within each bundle the collagen fibrils are both connected together and spaced apart by the chondroitin sulfate chains of type IX collagen (thin black lines). (b) With aging there is a loss of type IX collagen from the fibril surfaces. The loss of the type IX collagen from the fibril surfaces combined with an increased surface exposure of type II collagen results in aggregation of the collagen fibrils. Reproduced from Le Goff, M. M., and Bishop, P. N. (2008). Adult vitreous structure and postnatal changes. Eye 22: 1214–1222, with permission of the Nature Publishing Group.
there is a progressive loss of type IX collagen from the surface of the human vitreous collagen fibrils with aging, and that the half-life of type IX collagen on the fibril surfaces is just 11 years of age. This loss of type IX collagen (along with its chondroitin sulfate chains) will result in a loss of spacing between the collagen fibrils. Furthermore, the loss of type IX collagen results in the increased exposure of sticky type II collagen on the fibril surfaces, so when the collagen fibrils come into contact (as a result of eye movement) they will have a propensity to fuse irreversibly.
Weakening of the Vitreoretinal Adhesion In young eyes there is a relatively strong adhesion between the cortical vitreous and the ILL of the postoral retina, but this weakens with age. The cortical vitreous collagen fibrils do not generally insert directly into the postoral ILL, so it is assumed that the molecular basis of the vitreoretinal adhesion is interactions between components on the surface of cortical vitreous collagen fibrils and macromolecules on the inner surface of the ILL. There is evidence that type XVIII collagen is involved in vitreoretinal adhesion from studies of knockout mice and early PVD in patients with Knobloch syndrome, a rare autosomal recessive condition that results in a lack of type XVIII collagen. Type XVIII collagen is a heparan sulfate proteoglycan. It has been shown that opticin can bind heparan sulfates including those of type XVIII collagen and opticin and type XVIII collagen colocalize at the vitreoretinal interface. Therefore, the opticin on the surface of cortical vitreous collagen fibrils could bind heparan sulfate proteoglycans of the ILL including type XVIII collagen, thereby contributing to vitreoretinal adhesion. However, opticin knockout mice do not appear to develop spontaneous vitreoretinal disinsertion, suggesting that other molecular interactions also contribute to vitreoretinal adhesion, at least in the mouse eye.
Conclusions
Figure 7 The role of the chondroitin sulfate chains of type IX collagen in maintaining the spacing between vitreous collagen fibrils. Electron micrographs of vitreous collagen fibrils stained with uranyl acetate (to visualize the collagen fibrils) and the cationic dye Alcian blue (to visualize the chondroitin sulfate chains of type IX collagen). The Alcian blue-stained chondroitin sulfate chains appear to bridge between adjacent collagen fibrils and thereby both space the collagen fibrils apart and link them together. (Scale bar 300 nm).
Although the vitreous only contains a dilute dispersion of collagen fibrils, it is these fibrils that are central to the gel structure of the vitreous. Molecules on the surface of these collagen fibrils are essential for maintaining the gel state (such as type IX collagen) and play a key role in vitreoretinal adhesion. Alterations in the molecular interactions of the collagen fibrils lead to vitreous liquefaction, weakening of vitreoretinal adhesion, and a consequent predisposition to PVD. See also: Cellular Origin, Formation and Turnover of the Vitreous; Hereditary Vitreoretinopathies; Hyalocytes.
Molecular Composition of the Vitreous and Aging Changes
Further Reading Bishop, P. N. (2000). Structural macromolecules and supramolecular organisation of the vitreous gel. Progress in Retinal and Eye Research 19: 323–344. Bishop, P. N., Holmes, D. F., Kadler, K. E., McLeod, D., and Bos, K. J. (2004). Age-related changes on the surface of vitreous collagen fibrils. Investigative Ophthalmology and Visual Science 45: 1041–1046.
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Bos, K. J., Holmes, D. F., Meadows, R. S., et al. (2001). Collagen fibril organisation in mammalian vitreous by freeze etch/rotary shadowing electron microscopy. Micron 32: 301–306. Fukai, N., Eklund, L., Marneros, A. G., et al. (2002). Lack of collagen XVIII/endostatin results in eye abnormalities. EMBO Journal 21: 1535–1544. Wang, J., McLeod, D., Henson, D. B., and Bishop, P. N. (2003). Agedependent changes in the basal retinovitreal adhesion. Investigative Ophthalmology and Visual Science 44: 1793–1800.